The long-term goal of my laboratory is to understand how Hedgehog (Hh) signal is transduced to control a wild variety of cellular behaviors. Hh family of secreted proteins controls many aspects of animal development. Malfunction of Hh signaling has been linked to numerous human disorders including cancers. Hh family members exert their biological influence through an evolutionarily conserved, yet poorly defined signal transduction cascade. A critical step in Hh signal transduction is the activation of Cubitus interruptus (Ci), which is regulated by large protein complexes containing the kinesin-related protein Costal2 (Cos2). We have developed a novel method for detecting Cos2 interacting proteins in vivo and have identified several novel components of Cos2/Ci complexes. In the proposed study, we will use a combination of genetic, biochemical, cellular, and pharmacological approaches to investigate the mechanisms by which Cos2 and its interacting proteins regulate Ci phosphorylation, activity, and subcellular localization. In particular, we plan to 1) investigate the mechanism by which distinct Cos2 complexes regulate Ci nuclear translocation; 2) study the role and regulation of GSK3/Cos2 interaction in Ci phosphorylation; 3) investigate the role of casein kinase I (CKI) in Hh signaling and the underlying mechanism; 4) determine the role of a novel Cos2 interacting protein, Koro, and identify and characterize additional Cos2 interacting proteins that regulate Ci activity. Knowledge gained from the proposed study shall shed light into how Hh signal is transduced to control cell growth and patterning, and may provide new avenues for diagnosis and therapeutic intervention of cancers caused by mis-regulation of Hh signaling activity.